Charles Xavier

ETS Related Gene mediated Androgen Receptor Aggregation and Endoplasmic Reticulum Stress in Prostate Cancer Development

Mechanistic studies of deregulated ERG in prostate cancer and other cancers continue to enhance its role in cancer biology and its utility as a biomarker and therapeutic target. Here, we show that ERG, through its physical interaction with androgen receptor, induces AR aggregation and endoplasmic reticulum stress in the prostate glands of ERG transgenic mice. Histomorphological alterations and the expression of ER stress sensors Atf6, Ire1α, Perk, their downstream effectors Grp78/BiP and eIF2α in ERG transgenic mouse prostate glands indicate the presence of chronic ER stress. Transient activation of apoptotic cell death during early age correlated well with the differential regulation of ER stress sensors, in particular Perk. Epithelial cells derived from ERG transgenic mouse prostates have increased prostasphere formation with resistance to radiation induced cell death. Continued activation of cell survival factors, Atf6 and Ire1α during chronic ER stress due to presence of ERG in prostate epithelium induces survival pathways and provides a selection pressure in the continuum of ERG dependent neoplastic process. These novel insights will enhance the understanding of the mechanistic functions of ERG in prostate tumor biology and towards development of early targeted therapeutic strategies for prostate cancer.

Synergistic Activity with NOTCH Inhibition and Androgen Ablation in ERG-positive Prostate Cancer Cells

The oncogenic activation of the ETS-related gene (ERG) due to gene fusions is present in over half of prostate cancers in Western countries. Because of its high incidence and oncogenic role, ERG and components of ERG network have emerged as potential drug targets for prostate cancer. Utilizing gene expression datasets, from matched normal and prostate tumor epithelial cells, an association of NOTCH transcription factors with ERG expression status was identified, confirming that NOTCH factors are direct transcriptional targets of ERG. Inhibition of ERG in TMPRSS2-ERG–positive VCaP cells led to decreased levels of NOTCH1 and 2 proteins and downstream transcriptional targets and partially recapitulated the phenotypes associated with ERG inhibition. Regulation of NOTCH1 and 2 genes by ERG were also noted with ectopic ERG expression in LNCaP (ERG-negative prostate cancer) and RWPE-1 (benign prostate–derived immortalized) cells. Furthermore, inhibition of NOTCH by the small-molecule γ-secretase inhibitor 1, GSI-1, conferred an increased sensitivity to androgen receptor (AR) inhibitors (bicalutamide and enzalutamide) or the androgen biosynthesis inhibitor (abiraterone) in VCaP cells. Combined treatment with bicalutamide and GSI-1 showed strongest inhibition of AR, ERG, NOTCH1, NOTCH2, and PSA protein levels along with decreased cell growth, cell survival, and enhanced apoptosis. Intriguingly, this effect was not observed in ERG-negative prostate cancer cells or immortalized benign/normal prostate epithelial cells. These data underscore the synergy of AR and NOTCH inhibitors in reducing the growth of ERG-positive prostate cancer cells. Implications: Combinational targeting of NOTCH and AR signaling has therapeutic potential in advanced ERG-driven prostate cancers. Mol Cancer Res; 15(10); 1308–17. ©2017 AACR.

MP87-19 ETS RELATED GENE (ERG) DRIVEN ANDROGEN RECEPTOR AGGREGATION IS A KEY REGULATOR OF ENDOPLASMIC STRESS AND CELL SURVIVAL DURING PROSTATE CARCINOGENESIS

The goal of this study is to identify germline molecular determinants associating with ERG status of CaP. METHODS: Blood derived genomic DNA samples were prepared from 270 AA men and 129 CA men treated by radical prostatectomy at Walter Reed National Military Medical Center. ERG status was determined in whole mounted prostate specimens by immunohistochemistry (IHC) for ERG protein expression as a surrogate for the TMPRSS2-ERG fusion. Blinded blood samples were genotyped for SNPs on the Illumina Golden Gate platform using Infinium Oncoarray, a 500K genome wide BeadChip kit from Illumina. Data analysis approaches included association analyses based on logistic regression, Principal Component Analysis (PCA) and Efficient Mixed-Model Association eXpedited (EMMAX) analysis. Genotype imputation analysis is being performed by IMPUTE2 program. RESULTS: After applying rigorous sample and SNP QC steps on the datasets, SNP genotyping analysis was performed in 321 patients with 478,299 SNPs. Logistic regression, principal component analysis by EIGENSTRAT and a variance component approach, EMMAX analysis were performed to account for population structure. By EMMAX we identified SNPs associated with ERG status. The SNPs most significantly (<10-5) associated with ERG fusion status included rs6698333, an intron variant of Kruppel-like factor 17 (KLF17) and two SNPs (rs1889877, rs3798999) in the intron of adhesion G proteincoupled receptor B3 (ADGRB3). Fine-mapping of SNPs is underway by genotype imputation analysis (IMPUTE2) using the 1000 Genomes reference dataset, followed by independent validation. CONCLUSIONS: This study identified SNPs differentially associated with ERG status of CaP, a major driver oncogene in CaP. Although the biological significance as it relates to ERG status of CaP still needs to be determined, these SNPs, with independent validation, may help as markers in stratifying patients early (even before CaP is detected) for targeted prevention and treatment options.

MP87-14 STRUCTURE-ACTIVITY RELATIONSHIP STUDIES OF ERGI-USU, A HIGHLY SELECTIVE INHIBITOR FOR ERG POSITIVE PROSTATE CANCER CELLS

INTRODUCTION AND OBJECTIVES: While new prostate cancer (CaP) treatments (Abiraterone and Enzalutamide) have improved survival in castration resistant prostate cancer (CRPC), their benefits are short-lived and drug resistance develops likely due to numerous adaptive mutations. Accumulating evidence has established the androgen regulated TMPRSS2-ERG fusion as a common oncogenic driver that contributes to the early development and progression of over half of CaP. Therefore, ERG oncoprotein and ERG dependent pathways are promising targets for CaP therapy in early stages when cancer is most responsive to treatment. We previously identified a small molecule inhibitor, ERGi-USU, which selectively inhibits ERG protein and cell growth in ERG positive tumor cell lines and mouse xenograft models. In an effort to further develop ERGi-USU with enhanced efficacy we performed detailed structure-activity relationship (SAR) evaluation of ERGi-USU core structure and developed new derivatives. METHODS: Based on SAR of the core structure of ERGi-USU, 48 new derivatives were designed and synthesized by substitutions with alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or hydroxyl groups. The new ERGi-USU derivatives were evaluated for inhibition of cell growth and ERG protein levels in the TMPRSS2-ERG fusion harboring CaP cell line, VCaP. Four of these compounds have been selected for evaluation of ERG selectivity by defining IC50 in ERG positive malignant cells (VCaP, KG1, MOLT-4 and COLO320), ERG negative CaP cell line (LNCaP) or ERG positive normal primary endothelium-derived cells (HUVEC). RESULTS: Like parental compound, four new ERGi-USU derivatives exhibited inhibition of cell growth and ERG protein levels in ERG positive VCaP, KG1, MOLT-4 and COLO320 cell lines, with no or minimal effects on LNCaP and HUVEC cells. One of the new derivatives (ERGi-USU#6) showed increased efficacy for cell growth inhibition (IC501⁄40.074mM) compared to the parental ERGi-USU (IC501⁄40.200mM). Other three new compounds showed similar IC50 as the ERGi-USU. CONCLUSIONS: Comprehensive evaluation of ERGi-USU derivatives along with parental compound has continued to underscore selective inhibition of ERG positive tumor cells by these small molecules.

Abstract 5058: Silencing of NOTCH signaling enhances the sensitivity of ERG positive prostate cancer cells to AR inhibitors

Introduction: Androgen receptor (AR) signaling plays a critical role in all the stages of prostate cancer (CaP) ranging from organ confined to castration-resistant (CRPC) phases. Although androgen deprivation therapy (ADT) remains the mainstay treatment for advanced CaP, the inevitable transition from androgen- sensitive to CRCP presents the most significant challenge in CaP therapy. Androgen dependent expression of oncogenic ETS related gene (ERG) in half of all CaP in western countries plays critical role in the tumorigenesis of CaP through regulation of cancer specific signaling pathways. We found that NOTCH transcription factors are common targets of ERG in ERG positive cancer cells. NOTCH signaling pathway is an important signaling pathway in the development of drug-resistant tumor growth. In the current study we evaluated the combinatorial effects of NOTCH and AR inhibitors in the context of ERG positive prostate cancer cells. Methods: ERG, NOTCH1, NOTCH2 and downstream targets of NOTCH transcription factors were analyzed by Western blot assays. Dose and time kinetics of combining NOTCH inhibitor (γ-Secretase inhibitor 1, GSI-1) and AR inhibitors (Bicalutamide, Enzalutamide, and Abiraterone) were assessed in a panel of ERG positive or ERG negative CaP cells. Trypan blue exclusion, methylthiazole tetrazolium (MTT), or ApoTox-Glo™ Triplex assays were used to asses cell proliferation, apoptosis and drug cytotoxicity. Results: Prostate cancer cell lines with endogenous or ectopic expression of ERG showed upregulation of NOTCH1 and NOTCH2. The NOTCH inhibitor, GSI-1 conferred an increased sensitivity to all tested AR inhibitors (Bicalutamide, Enzalutamide, and Abiraterone) with bicalutamide showing the most robust inhibition of AR, ERG, NOTCH1, NOTCH2, PSA, decreased cell growth and enhanced apoptosis in ERG positive VCaP cells. This observation was not seen in ERG negative LNCaP cells or in ERG positive primary endothelial cells. Conclusions: NOTCH inhibitor enhanced sensitivity of AR inhibitors in ERG positive VCaP cells growth. The combination of the GSI-1 with AR inhibitors has shown synergistic effect when compared to single agent treatment. Taken together, our study suggests that NOTCH inhibitors may enhance the actions of AR inhibitors in the treatment of ERG positive prostate cancers. Inhibition of AR and NOTCH signaling may offer new opportunities in assessing ERG targeted therapy for prostate cancer. Citation Format: Ahmed A. Mohamed, Shyh-Han Tan, Shilpa Katta, Charles P. Xavier, Lakshmi Ravindranath, Wei Huang, Hua Li, Meera Srivastava, Shashwat Sharad, Taduru Sreenath, Gyorgy Petrovics, Albert Dobi, Shiv Srivastava. Silencing of NOTCH signaling enhances the sensitivity of ERG positive prostate cancer cells to AR inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5058. doi:10.1158/1538-7445.AM2015-5058